Designing neuronal tissue constructs that mimic brain-specific architecture Objective(s): This proposal develops an in vivo approach for repairing damaged cerebral cortex circuits with engineered neural tissue. Specifically, the proposed studies seek to generate transplantable constructs with attributes of grey and white matter structure to optimize their match to normal brain architecture. Upon transplantation, these constructs will be evaluated for their survival and integration into host brains. Emphasis will be placed on obtaining evidence of functional recovery in transplanted animals. In addition, this proposal will obtain proof-of-principle data that functional neural tissue can be generated from human stem cell sources, a crucial step toward translating this work into a clinically viable therapy. Research Design & Methodology: This proposal builds upon recent advances in tissue engineering to generate transplantable neural tissue that replicate various aspects of grey and white matter architecture. We hypothesize that recreating brain-specific architecture is vital to properly repairing brain circuits and restoring brain function. There will be three phases to this work. Glutamatergic cortical neurons will be differentiated from human stem cell sources. The phenotype and electrophysiology of the differentiated neurons will be assessed, and three-dimensional cultures of these cells will be grown. In parallel, two types of constructs will be engineered from rat embryonic and then human stem cell-derived neurons. The first will be a multi-layered construct that reflects cortical architecture. Two scaffold layers will be stacked on each other, one with neurons from layers 2-4 and the other with neurons from layers 5-6. Neurons from each layer will be transduced with different opsins for layer-specific stimulation. The second construct will be transplantable 3D tracts of axons using previously developed mechanical elongation methods. The survival and network activity of each construct will be carefully analyzed. Multi-layered constructs will be transplanted into primary motor cortex in uninjured and injured (lateral fluid percussion) rat brains. Construct survival will be determined with immunohistochemistry at 2 weeks and 1, 2, and 6 months after transplantation. Functional integration will be assessed at these time points by optically stimulating the construct and recording muscle activity and visual evidence of movement in the extremities. Motor function will be evaluated using the rotarod and beam walk tasks, as well as assessments of locomotion speed and distance. Axonal tissue will be transplanted across the two hemispheres of the rat brain with an intact or disconnected corpus callosum. Construct survival will be evaluated as above. Functional integration will be ascertained by stimulating one hemisphere of the host brain and recording from the contralateral side. Motor coordination will be assessed through running performance on a wheel with uneven rungs. We will also evaluate the cognitive performance of transplanted animals to determine if the transplantation procedure has any detrimental effects. Findings: This is a new proposal. Clinical Relationships: Traumatic brain injury (TBI) is the signature injury of U.S. military personnel involved in recent conflicts in Iraq and Afghanistan and results in a wide range of disabling sequelae. Replacing lost neural tissue is important for restoring neurological function and enhancing the effects of complementary neuromodulation and plasticity-based therapies.